Tumor necrosis factor-alpha (TNF-α or TNF) is cytokine which is a member of acute-phase proteins involved in an inflammatory response. TNF-α is mainly secreted by activated macrophages, and is also secreted in a variety of cells such as helper T cells, NK cells, neurons, and the like.
Immune responses of TNF are causes of various diseases (rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa and refractory asthma).
Accordingly, TNF inhibitors are often used to treat diseases caused by TNF.
TNF inhibitor protein pharmaceuticals include infliximab (product: Remicade), adalimumab (product: Humira), certolizumab pegol (product name: Cimzia) as monoclonal antibody proteins, and etanercept (product name; Enbrel) antibody as circulating receptor fusion protein.
Production amount of the antibody protein pharmaceuticals has increased in accordance with the development of recombinant technology, cell culture techniques, and purification techniques. However, the antibody protein requires appropriate formulations thereof due to physical and chemical instability caused by a large molecular weight and a complex structure as compared to general protein pharmaceuticals.
The antibody protein has a problem of forming aggregation which is representative phenomenon generated by physical instability. In fact, factors causing aggregation are variously present in purification, formulation, storage processes of the antibody protein. For example, the aggregation may be caused by at least one of the following factors. In purification, pH, salt type, salt concentration, temperature, contact with air, stirring speed, and the like, that are not optimum conditions, affect the aggregation, and in formulation, protein concentration conditions may affect the aggregation. Further, in changing buffers, passing through the filter, stirring, and the like, may affect the aggregation, and in storing, temperature change, pH change, contact with air, stirring, and the like, may affect the aggregation. Besides, the aggregation may occur even when the formulation including the protein is exposed to light. Because when exposed to light, a photoreactive material is produced and combined with other groups to cause variants. In addition, packing container may also be a cause of aggregation. The reason is because trace amount of metal ions exposed from the packing material to the formulation may promote hydrolysis of amide bonds (Current Pharmaceutical Biotechnology, 2009, 10, 348-351, Hamada, H. et al.).
As described above, the aggregation phenomenon may be caused by various factors including environmental factors such as temperature, pH, buffer type, concentration, ionic strength, excipient type, concentration of protein, the presence or absence of reducing materials, the presence of impurities and the state of a container, or purification methods, and the like. However, the aggregation phenomenon shown in the antibody protein is mainly caused by water solubility decreased when hydrophobic regions of the antibody protein are exposed due to structural change. That is, the hydrophobic regions of the protein molecules are assembled to form massive aggregation, wherein the formed aggregation may progress in an irreversible form due to covalent bonding generated between molecules in the antibody protein (Current Pharmaceutical Biotechnology 10(2009), 348-351, Hamada, H. et al.).
The antibody protein aggregated by the above-described reasons generally has reduced activities or loses activities over time. In addition, when these aggregated proteins are administered to a human body, the protein has antigenicity which is not exhibited in a non-aggregation state, which may induce production of an antibody (anti-drug antibody, ADA). Therefore, it is required to develop a method of reducing the aggregation of protein and stabilized formulation of protein having a reduced level of aggregation (Current Trends in Monoclonal Antibody Development and Manufacturing Biotechnology: Pharmaceutical Aspects Volume XI, 2010, pp 271-291).
Under such technical background, the present inventors found that basic amino acid, sugar and/or ammonium salt is capable of inhibiting aggregation of TNFα binding protein, and increasing stability of the protein so as to be easily preserved and stored for a long period of time, and completed the present invention.